This application is a part of the competitive renewal for the Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD) in response to RFA-AA-17-012. Pathological outcomes of Fetal Alcohol Spectrum Disorder (FASD) stemming from prenatal alcohol exposure (PAE) are devastating and highly variable, especially in regards to cognitive and learning deficits apparent in later life. Early intervention for such deficits is imperative for optimal outcomes; however, the pattern and magnitude of these deficits are not predictive even when accounting for the level of alcohol exposure, which by itself is difficult to accurately assess. Therefore, early, and precise biomarkers for predicting the risk of cognitive and behavior problems are crucial for establishing an effective treatment. This project aims at establishing a novel approach to identifying such biomarkers for predicting the risk of children afflicted with FASD. Based on our preliminary data, we hypothesize that single-cell level epigenetic changes detectable in blood cell samples serve as biomarkers in predicting risks of cognitive and learning deficits before their symptomatic manifestations. By employing cutting-edge cellular droplet technology, we will test this hypothesis using the mouse model of PAE (Aim 1), and examine whether these biomarkers are applicable for human patients with a history of PAE (Aim 2). The Hashimoto-Torii lab will perform the single-cell droplet digital PCR- based biomarker analyses (drop-PCR) with both human and mouse blood samples. The Torii lab will collect the mouse blood samples, perform comprehensive mouse behavior analyses, and statistically evaluate potential correlations between the animal behaviors and drop-PCR results. The Chambers lab will collect the human blood samples, perform neurocognitive tests, and statistically evaluation of potential correlations between these test scores and the drop-PCR results. This project will allow for critical assessment in linking biomarkers with comprehensive evaluations of neurocognitive deficits, brain structural abnormalities and facial dysmorphology. In addition, these studies maximize the potential our collaborations with other CIFASD research including the neurobehavioral (Chambers), genetic (Foroud) and dysmorphology core (Jones) projects. Cross-sectional approaches using controlled animal studies (Eberhart and Parnell) will provide essential mechanistic insights. Our identified biomarkers and those obtained through studies using cytokine (Chambers) and miRNA (Weinberg) panels generated for the same PAE patients will provide a rare opportunity to test this combined biomarker strategy for accurate prediction of PAE outcomes. By capitalizing on CIFASD infrastructure, this project will develop innovative single-cell biomarkers that impact FASD research and translational science at large.